Selection of candidate reference genes and validation for real-time PCR studies in rice plants exposed to low temperatures.

Rice is a cereal that presents a great ability to adapt to different soil and climate conditions. However, as it is a tropical crop with C3 metabolism, it performs better in warm temperatures with high solar radiation. Tolerance to stress caused by low temperatures is a highly complex process that involves various metabolic pathways and cellular compartments, resulting in general or specific effects on plant growth and development. In order to observe the true effect of a particular stress on genetic expression, reference genes need to be chosen for real-time PCRs, the expression levels of which should remain stable independent of the situation imposed. In this paper, the expression stability was evaluated of the actin 11 (ACT11), ubiquitin-conjugating enzyme 2 (UBC-E2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta tubilin (ß-Tubulin), eukaryotic initiation factor 4α (eIF-4-α), eukaryotic initiation factor 1α (eIF-1-α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin (TIP41), and cyclophilin genes, in two rice genotypes cultivated in low temperature (13°C) conditions in vegetative stage (V4). The analysis material (leaves) was collected after 0, 6, 24, 48, and 72 h of exposure to the stress. In this study, the geNorm, BestKeeper, ΔCt, NormFinder, and RefFinder methods were used to evaluate the expression stability of the candidate reference genes. The results revealed that the most indicated genes for all the analysis methods were UBQ10 and UBQ5 for BRS Bojuru and BRS Pampa, respectively. On the other hand, the eIF-1-α gene presents the least expression stability and is not indicated for studies of rice plants subjected to low temperatures. The validation with the antioxidant system genes SODCc1-Cu/Zn, CATC, APX2, and GR2 confirmed the importance of using previously tested normalizing genes for adequate real-time PCR results.

Changes in gene expression and catalase activity in Oryza sativa L. under abiotic stress.

Different rice (Oryza sativa L.) genotypes were subjected to high salinity and low temperature (150 mM NaCl and 13°C, respectively) for 0, 6, 24, 48, or 72 h. We evaluated the simultaneous expression of the genes OsCATA, OsCATB, and OsCATC, correlated gene expression with enzyme activity, and verified the regulation of these genes through identification of cis-elements in the promoter region. The hydrogen peroxide content increased in a tolerant genotype and decreased in a sensitive genotype under both stress conditions. Lipid peroxidation increased in the tolerant genotype when exposed to cold, and in the sensitive genotype when exposed to high salinity. Catalase activity significantly increased in both genotypes when subjected to 13°C. In the tolerant genotype, OsCATA and OsCATB were the most responsive to high salinity and cold, while in the sensitive genotype, OsCATA and OsCATC responded positively to saline stress, as did OsCATA and OsCATB to low temperature. Cis-element analysis identified different regulatory sequences in the catalase promoter region of each genotype. The sensitive genotype maintained a better balance between hydrogen oxyacid levels, catalase activity, and lipid peroxidation under low temperature than the resistant genotype. OsCATA and OsCATB were the most responsive in the salt-tolerant genotype to cold, OsCATA and OsCATC were the most responsive to saline stress, and OsCATA and OsCATB were the most responsive to chilling stress in the sensitive genotype. There were positive correlations between catalase activity and OsCATB expression in the tolerant genotype under saline stress and in the sensitive genotype under cold stress.

Expression of LTP genes in response to saline stress in rice seedlings.

Saline stress is one of the primary factors limiting increased rice productivity in the southern region of Brazil. Farming can be affected by salinity that is due to both the origin of the soils as well as the irrigation water. Lipid transfer proteins (LTPs) have many physiological functions, including in the response to saline stress. Therefore, the objective of this study was to quantify the relative expression of 11 genetic isoforms that encode LTP1-type proteins in rice genotypes tolerant and sensitive to saline stress in the vegetative period. When the plants reached development stage V4, alternating irrigation was started with nutritive solution and water containing 150 mM NaCl. The LTP7 gene showed an increase in expression by 13.81-fold after 96 h of stress exposure in the saline-tolerant group, whereas the LTP10 gene expression level was increased by 71.10-fold after 96 h in the saline-sensitive group. The LTP26, LTP23, and LTP18 genes showed increased expression in both genotypes; however, the expression levels and response times were different. Thus, LTP7 and LTP10 showed the highest response to salinity. The LTP18, LTP23, and LTP26 genes were negatively correlated with the response to salinity.

Evaluation of reference genes for RT-qPCR studies in the leaves of rice seedlings under salt stress.

To obtain accurate and reliable results for the expression of genes of interest using quantitative real-time polymerase chain reaction (RT-qPCR) techniques, it is necessary to normalize the data by comparing them to constitutive genes that exhibit uniform expression levels under experimental conditions. In this study, the stability of expression was evaluated for the following ten candidate reference genes in rice leaves (Oryza sativa L.) from the BRS Bojuru and BRS Ligeirinho genotypes that were subjected to salt stress (150 mM): actin 11 (ACT11), beta-tubulin (ß-TUB), eukaryote elongation factor 1-α (Eef-1), eukaryotic initiation factor 4-α (eIF-4-α), E2 ubiquitin-conjugating enzyme (UBC-E2), ubiquitin 5 (UBQ5), ubiquitin 10 (UBQ10), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TIP41-like, and cyclophilin. The stability of expression for the aforementioned genes was then compared to that of three LTP genes using UBQ10, Eef-1, and eIF-4-α as references. After analyzing the expression levels using analysis of variance tests, the results indicated that UBQ10 was the most stable in all treatments (M = 0.404 and SV = 0.327). Furthermore, the eIF-4-α, TIP41-like, and cyclophilin genes exhibited the highest total coefficient of variation (CV = 269, 169.2, 179.2, respectively), which signifies that they exhibited the least stable expression. The expression levels of each candidate gene (LTP7, LTP10, and LTP13) were in contrast to the reference genes. Therefore, we concluded that UBQ10 is the best reference gene for RT-qPCR reactions under the experimental conditions. The expression analysis of LTP7, LTP10, and LTP13 confirmed the importance of validating reference genes to achieve accurate RT-qPCR results.

Composição do óleo essencial de quatro espécies do gênero Plectranthus / Essential oil composition of four Plectranthus species

O gênero Plectranthus é considerado um dos mais ricos em óleos essenciais dentro da família Lamiaceae, compreendendo muitas espécies com propriedades medicinais. Algumas destas são conhecidas popularmente como boldo, as quais possuem semelhanças taxonômicas e diversas sinonímias, possuindo ações anti-dispépticas, analgésicas e digestivas. O objetivo deste estudo foi avaliar quantitativa e qualitativamente os óleos essenciais presentes nas folhas das espécies P. amboinicus, P. barbatus, P. grandis e P. neochilus. A extração do óleo foi realizada por hidrodestilação, utilizando pentano como solvente extrator, repetida por três vezes para cada uma das espécies. A análise dos componentes dos óleos essenciais das quatro espécies de Plectranthus, através da CG/EM, permitiu identificar 14 componentes químicos, sendo a maioria sesquiterpenos. O trans-cariofileno se apresentou em elevada concentração nos óleos estudados. Alguns componentes químicos demonstraram ser específicos para cada espécie e outros apresentaram ocorrência comum a todas as quatro, possibilitando a diferenciação das mesmas em dois grupos, um formado por P. amboinicus e P. neochilus e o outro por P. grandis e P. barbatus. Conclui-se que as quatro espécies de boldo apresentam diferenças significativas quanto ao teor e à constituição química do óleo essencial.

Plectranthus has been considered one of the richest genera in essential oils within the Lamiaceae family, which includes several species with medicinal properties. Some of them are commonly known as boldo and present taxonomic similarities and several synonymies, with antidyspeptic, analgesic and digestive actions. The aim of this study was to evaluate quantitatively and qualitatively the essential oils from P. amboinicus, P. barbatus, P. grandis and P. neochilus leaves. The oil was extracted by hydrodistillation using pentane as extracting solvent and was repeated three times for each species. The analysis of essential oil components by GC/MS in the four Plectranthus species identified 14 chemical components, mostly sesquiterpenes. High concentration of trans-caryophyllene was found in the studied oils. Some chemical components were specific for each species and other components had common occurrence in all four species, allowing their differentiation into two groups, one composed of P. amboinicus and P. neochilus and another one by P. grandis and P. barbatus. In conclusion, the four boldo species had significant differences as to essential oil yield and chemical composition.

Diversidade genética entre acessos de espinheira-santa (Maytenus ilicifolia Mart. ex Reis.) coletados no estado do Rio Grande do Sul, Brasil / Genetic diversity of "espinheira-santa" (Maytenus ilicifolia Mart. ex Reis.) accessions collected in Rio Grande do Sul State, Brazil

Maytenus ilicifolia Mart. ex Reis., popularmente conhecida como espinheira-santa, é espécie autóctone pertencente à família Celastraceae, usada para tratamento de úlceras gástricas e gastrites. Devido à importância medicinal, houve aumento no extrativismo das populações naturais, tornando-a uma espécie prioritária para a conservação, a fim de evitar a erosão genética. Buscou-se com este trabalho analisar a diversidade genética de 20 acessos de M. ilicifolia coletados em diferentes localidades no Rio Grande do Sul. Utilizando marcadores moleculares do tipo AFLP, foram testadas oito combinações de primers, que geraram 455 bandas eletroforéticas, 100 por cento polimórficas. As combinações de primers E-ACC/M-CAA, E-ACG/M-CTA, E-ACG/M-CTC apresentaram o maior número de bandas eletroforéticas, 71 cada, totalizando 46,80 por cento do polimorfismo total. Os valores de similaridade genética calculada pelo coeficiente simple matching foram utilizados para gerar o dendrograma de similaridade pelo método UPGMA. Foi obtido alto coeficiente de correlação cofenética (r=0,94), demonstrando elevada representatividade dos dados de similaridade genética no dendrograma. Pela AMOVA verificou-se que 89,33 por cento da diversidade total ocorreram entre indivíduos dentro das populações. A caracterização molecular de acessos de Maytenus ilicifolia por meio de AFLP foi eficiente para identificar diversidade genética. Através da análise de similaridade genética o banco de germoplasma poderia ser composto com os acessos que apresentaram menor similaridade e maior número de alelos, permitindo com que estes fornecessem ampla cobertura do genoma que compõem Maytenus ilicifolia. Os acessos que ficaram agrupados em mesmo cluster e com número reduzido de alelos podem ser descartados deste banco. A diversidade genética intrapopulacional identificada por esse marcador foi muito maior do que aquela entre populações.

Maytenus ilicifolia Mart. ex Reis., popularly known as "espinheira-santa", is an autochthonous species belonging to the Celastraceae family. This species is used to treat ulcers and gastritis. Due to its medicinal importance, the exploitation of natural populations has increased, making the conservation of this species essential to prevent genetic erosion. The aim of this work was to analyze the genetic diversity of 20 M. ilicifolia accessions collected in different localities of Rio Grande do Sul State. Using AFLP-type molecular markers, eight primer combinations were tested, producing 455 electrophoretic profiles, with 100 percent polymorphism. The primer combinations E-ACC/M-CAA, E-ACG/M-CTA and E-ACG/M-CTC presented the largest number of electrophoretic profiles, 71 each, totaling 46.80 percent of the total polymorphism. The values of genetic similarity estimated by Simple Matching Coefficient were used to produce the dendrogram of similarity by the UPGMA method. A high cophenetic correlation coefficient (r = 0.94) was obtained, demonstrating high representativeness of the data of genetic similarity in the dendrogram. Using AMOVA, 89.33 percent of the total diversity were observed among individuals from the same population. The molecular characterization of Maytenus ilicifolia accessions by AFLP allowed the identification of genetic diversity. The genetic similarity analysis indicated that the germplasm bank could be composed of the accessions presenting the lowest similarity and the largest numbers of alleles, providing a comprehensive coverage of Maytenus ilicifolia genome. The accessions that were grouped into one same cluster and with a reduced number of alleles could be disposed of this bank. The genetic diversity identified by this marker within populations was much greater than that between populations.